RNA interference (RNAi) is a powerful tool for performing loss of function studies in diverse organisms by transiently shutting of gene expression. Various methods have been developed to allow for efficient RNAi.
One of the common approaches is to use small inhibitory (siRNA) molecules which are typically double-stranded RNA molecules of a length of 15 to 30 nucleotides. The sequences of such siRNA molecules are selected so that they match sequences of the mRNA to be silenced by RNAi. The siRNAs are then brought into contact with the organism or cell for which gene silencing studies are to be performed. The siRNA molecules are integrated into the RISC complex a complex enzymatic machinery involving the so-called Ago proteins mediating the separation of the double-stranded siRNA molecules and effecting the hybridization of single stranded siRNA molecules with the target mRNA ultimately leading to the desired transient silencing of the respective mRNA, i.e. gene that is targeted by the siRNAs.
RNAi and siRNAs can be used in a versatile manner. For example, siRNA libraries may be designed to perform screens for loss of function studies addressing not only one, but numerous and, in principle, all genes of a particular cell or organism.
Some of the drawbacks of RNAi and siRNAs, which have been increasingly recognised in recent years, are so-called off-target-effects and efficacy as well as problems relating to the manufacturing of pools of siRNAs, which either allow for silencing of the expression of various genes at the same time, or a single gene by using a multitude of siRNAs being specific for that specific target gene.
In principle, the selectivity of RNAi can be addressed by properly selecting siRNAs. For example, one may select the sequence of an siRNA that determines which sequence of the gene to be silenced will be recognised such that a sequence is selected that should in principle be unique to the target gene and not be found in other sequences. By properly selecting such siRNA sequences, it should be possible to ensure that only the gene of interest is silenced. However, even though it should in principle be possible to select target siRNA sequences such that no other target sequences are recognised by the siRNAs with the consequence that no off-target-effects should occur, such siRNAs are not necessarily effective to the desired degree.
Effectiveness is determined inter alia by the fact that a target sequence may not easily be accessible in the in vivo situation to the siRNA due to interaction with proteins within a cell or the fact that the sequence of the target genes may adopt confirmations that render them non- or at least partially accessible to the siRNA. Due to these facts, an siRNA sequence which according to common selection procedures should not provide any off-target effects, may not prove effective or may also impact the expression of other genes. In view of the aforementioned problems, it may be necessary to design siRNAs with different sequences and to use them simultaneously in order to silence the expression of a single gene. For reasons not understood using numerous siRNA sequences against the same target gene, the danger of off-target effects may be reduced, perhaps by increasing the signal to nose ratio for specific siRNAs over non-specific or non-effective siRNAs.
Manufacturing of siRNAs by e.g. solid phase chemistry can be rather time and cost consuming. Particularly if one wants to produce complex pools of siRNAs either of siRNAs being directed to the same target gene or siRNA pools recognising different target genes can thus become prohibitive from a cost perspective.
Nevertheless such pools of siRNA sequences are of high interest because, as mentioned before, they allow efficient silencing of the expression of a single gene as then not each and every siRNA has to be tested stepwise. Rather, one can quite straigthforwardly silence a gene by using such a pool or one can even silence numerous genes at the same time.
In the light of this background, there is thus continuing interest in methods that allow for provision of pools of siRNAs that allow silencing of either expression of a single gene and/or that allow silencing of expression of numerous genes at the same time. Furthermore, there is a continuing interest in providing new methods for efficiently producing siRNAs and in particular the aforementioned siRNA pools. It is inter alia these problems that the present invention addresses.